1. Field of the Invention
The present invention relates to the use of nitric oxide to treat hemoglobin-containing erythrocytes in a diluent, including whole blood or at least one blood component, as well as compositions and methods of making and using thereof.
2. Related Background Art
Similarities Between Carbon Monoxide and Nitric Oxide
Carbon monoxide, a well-known environmental pollutant, has a high affinity for hemoglobin (Hb) and causes CO poisoning by binding to the heme groups of Hb in competition with O.sub.2. Its binding not only reduces the O.sub.2- binding capacity of Hb but also modifies Hb toward a high-affinity state rendering Hb incapable of delivering O.sub.2 to peripheral tissues effectively. Furthermore, the so-formed CO-bound Hb is highly stable and its toxic effect on the mammalian physiology is persistent and cumulative. The U.S. Environmental Protection Agency specifies the occupational limits of CO to be 8 ppm for 9-hour, and 35 ppm for 1-hour, inhalation for the adult, respectively (The U.S. Environmental Protection Agency, Publication AP-84 (1971)). Additionally, nitric oxide (also called nitrogen monoxide or NO) has more than a thousand-fold higher affinity for Hb than CO (Gibson, Q. H., and Roughton, F. J. W., Proc. Roy. Soc. London B. Biol. Sci. 163:197-205 (1965)). NO was thus expected to cause blood poisoning by reducing O.sub.2 binding and delivery capacities as does CO, and thus be toxic if present in more than very low concentrations in the blood.
Physiological Effects of Nitric Oxide
While nitric oxide is produced in vivo by several different forms of NO synthases (NOS), it is present in very low concentrations in the blood (generally less than one micromolar). When present in the blood, as it is an uncharged diatomic molecule with limited stability, NO can readily penetrate through cellular structures and act as a paracrine on its primary target, soluble guanylyl cyclase, in local environments immediately after its production. Nitric oxide activates soluble guanylyl cyclase to produce cyclic guanosine monophosphate (cGMP) which, in turn, initiates cGMP-dependent cascade reactions, leading to a wide range of biochemical, cellular, and physiological responses.
Such responses result from a low steady-state concentration of NO (&lt;1 .mu.M) in the blood, which involve maintaining normal vascular tone and other necessary conditions for the systemic and pulmonary circulation. A delicate balance exists between production of NO by NOSs and sequestration of NO, primarily by Hb in the erythrocyte, that maintains the homeostasis of the NO concentration in the plasma. The concentration of NO in the plasma is sometimes altered during infection and inflammation and by administration of NOS inhibitors or NO-generating reagents, such as nitroglycerin and nitrite. Such changes can wreak havoc in the circulatory system. Constriction of blood vessels, resulting in elevated blood pressure, vascular adhesion of leukocytes, and aggregation of platelets have been observed when the concentration of NO is increased. Free NO in the plasma constantly diffuses into the erythrocyte and immediately reacts with Hb, which acts as a NO scavenger. Stoichiometric reactions of NO with oxyHb are apparently fast and have been used for a spectrophotometric assay of NO (Doyle, M. P., and Hoekstra, J. W., J. Inorg. Chem. 14:351-358 (1981)). Thus, it has been generally assumed that free NO in the blood is scavenged by rapid reaction with oxyHb to produce such bio-inactive products as metHb and nitrate under physiological conditions. The so-formed metHb is recycled back to bioactive deoxyHb by metHb reductase in the erythrocyte.
Kosaka and Seiyama reported that the O.sub.2 binding curve of the blood of the rat treated with nitroglycerin was approximately 10 torr right-shifted (Kosaka, H. and Seiyama. A., Biochem. Biophys. Res. Commun. 218:749-752 (1996)) and observed increases in O.sub.2 delivery in hepatic sinusoids in nitroglycerin-treated rats (Kosaka. H. and Seiyama, A., Nature Science 3:456-459 (1997)).
Addition of small quantities of nitric oxide (NO) to hemoglobin (Hb) in solution (Hille, R., et al., J. Biol. Chem. 254:12110-12120 (1979)) and in the erythrocyte (Kosaka, H., et al., Am. J. Physiol. 266:C1400-C1405 (1994)); Erikson, L. E. G., Biochem. Biophys. Res. Commun. 203:176-181 (1994) converts a small fraction of the total Hb molecules to .alpha.-nitrosylHb. Intravenous administration of nitroglycerin, nitrite, and some cytokines to rats partially converts Hb to .alpha.-nitrosylHb (Kosaka, H. and Seiyama. A., Biochem. Biophys. Res. Commun. 218:749-752 (1996); Kosaka. H. and Seiyama, A., Nature Science 3:456-459 (1997)). The maximal fraction of .alpha.-nitrosylHb produced in rats is, however, less than 2% of the total Hb in rats (Kosaka, H., et al., Am. J. Physiol. 266:C1400-C1405 (1994)); Erikson, L. E. G., Biochem. Biophys. Res. Commun. 203:176-181 (1994); Kosaka, H. and Seiyama. A., Biochem. Biophys. Res. Commun. 218:749-752 (1996); Kosaka. H. and Seiyama, A., Nature Science 3:456-459 (1997)).
Further addition of these NO-producing compounds results in increasing amounts of tetra-nitrosylHB and metHb, both of which have no ability to transport O.sub.2 and thus are toxic (Kosaka, H., et al., Am. J. Physiol. 266:C1400-C1405 (1994)); Erikson, L. E. G., Biochem. Biophys. Res. Cornmun. 203:176-181 (1994); Kosaka, H. and Seiyama. A., Biochem. Biophys. Res. Commun. 218:749-752 (1996); Kosaka. H. and Seiyama, A., Nature Science 3:456-459 (1997)).
The only way to produce substantially pure .alpha.-nitrosylHb has been to physically separate Hb into .alpha.- and .beta.-subunits, expose the isolated .alpha.-subunits to NO to produce .alpha.-nitrosyl subunits, and then recombine the isolated .beta.-subunits to regenerate a tetrameric .alpha.-nitrosylHb, in which only .alpha.-subunits contain NO: .alpha.(Fe--NO).sub.2 .beta.(Fe).sub.2. .alpha.-nitrosylHb was reported to be a low-affinity O.sub.2 carrier. Yonetani, T., Proc. Japanese Medical Soc. Magn. Reson, (1995); Yonetani T., (Abstract 106S), Proc. 35th ESR Discussion Conference, Yamagata, Japan (1996), p. 15).
The Problem of Expired Blood in Blood Banks
Huge amounts of blood and blood products at blood banks are discarded after certain periods of storage (such as 2-3 weeks), due to expiration of the blood and blood products. This is because the concentration of both bisphosphoglycerate (BPG), a natural allosteric effecter, and the pH inside of the erythrocyte, decrease during storage and consequently the oxygen affinity of Hb increases due to conversion to the high affinity form, rendering the stored blood ineffective for transfusion due to low oxygen delivering capacity. As BPG is impermeable to the erythrocyte membrane, external administration of BPG to blood cannot restore its oxygen delivering capacity.
U.S. Pat. No. 5,122,539 (issued Jun. 16, 1992) describes a new allosteric effecter, which decreases oxygen affinity of hemoglobin. Its structure is unrelated to 2,3-BPG, the natural allosteric effecter of the erythrocyte, but its function is similar. However, due to its chemical structure, this compound is expected to not pass through the erythrocyte membrane, such that this method will not have any practical applicability for treating blood or blood products to increase oxygen delivery.
Accordingly, there is a long-felt need to provide methods and compositions that counteract the loss of BPG from stored blood or blood components, in order to increase its oxygen delivering capacity so that it can be used for longer periods of time and will not have to be discarded, usually at about three weeks after the storage is begun.
Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentability of any of the claims of the present application. All statements as to the date or representation of the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.